1. Field of the Invention
The invention relates to antisense oligonucleotides. More particularly, the invention relates to oligonucleotides having modified internucleotide linkages that render the oligonucleotides more resistant to nucleolytic degradation.
2. Summary of the Related Art
Synthetic oligonucleotides have become important tools in basic scientific research. Recently, synthetic oligonucleotides have been successfully used in the area of regulation of gene expression, which has laid the foundation for a novel therapeutic approach, known as antisense oligonucleotide therapy, for the treatment of various virus infections and disorders of gene expression. Several investigators have demonstrated the ability of oligonucleotides to inhibit virus propagation and to modulate gene expression in vitro.
Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 75: 285-288 (1978) discloses specific inhibition of Rous Sarcoma Virus replication in infected chicken fibroblasts by a 13-mer synthetic oligodeoxynucleotide that is complementary to part of the viral genome.
Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146 (1986) discloses inhibition of replication and expression of human immunodeficiency virus (HIV-1, then called HTLV-III) in cultured cells by synthetic oligonucleotide phosphodiesters complementary to viral RNA.
Recent studies have shown that oligonucleotides act with greater efficacy in the antisense approach when the oligonucleotides are modified to contain artificial internucleotide linkages that render the oligonucleotides resistant to nucleolytic degradation. These studies have involved the use of a variety of artificial internucleotide linkages. The most well studied artificial internucleotide linkages have been methylphosphonate, phosphorothioate and various phosphoramidate internucleotide linkages.
Sarin et al., Proc. Natl. Acad. Sci. USA 85: 7448-7451 (1988) teaches that oligodeoxynucleoside methylphosphonates are more active as inhibitors of HIV-1 than conventional oligodeoxynucleotides.
Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7083 (1988) teaches that oligonucleotide phosphorothioate and various oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional oligodeoxynucleotides.
Agrawal et al., Proc. Natl. Acad. Sci. USA 86: 7790-7794 (1989) discloses the advantage oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
Gao et al., Antimicrob. Agents and Chem. 34: 808 (1990) discloses inhibition of HSV by oligonucleotide phosphorothioates.
Storey et al., Nucleic Acids Res. 19: 4109 (1991) discloses inhibition of HPV by oligonucleotide phosphorothioates.
Leiter et al., Proc. Natl. Acad. Sci. USA 87: 3430 (1990) discloses inhibition of influenza virus by oligonucleotide phosphorothioates.
Unfortunately, oligonucleotide phosphorothioates increase resistance to nucleolytic degradation but do not provide complete resistance in vivo.
Agrawal et al., Proc. Natl. Acad. Sci. USA 88: 7595-7599 (1991) teaches that oligonucleotide phosphorothioates are extensively degraded from the 3' end in mice.
The greater efficacy in the antisense approach of modified oligonucleotides having artificial internucleotide linkages that render the oligonucleotides resistant to nucleolytic degradation underscores the importance of developing oligonucleotides having new artificial internucleotide linkages that provide even greater resistance to nucleolytic degradation. Non-ionic oligonucleotides are of particular interest, because of their improved uptake by cells. A possible candidate as a new and useful non-ionic artificial internucleotide linkage is the alkylphosphonothioate linkage. However, no procedure has been developed to allow the incorporation of alkylphosphonothioate internucleotide linkages into synthetic oligonucleotides. Previous attempts have been limited to solution phase synthetic efforts to produce dinucleotides containing a methylphosphonothioate internucleotide linkage.
Brill and Caruthers, Tet. Lett. 28: 3205-3208 (1987) and Tet. Lett. 29: 1227-1230 (1988) disclose an approach using methyl phosphonothioic dichloride to produce dinucleotides having a methylphosphonothioate internucleotide linkage in 56% yield.
Roelen et al., Nucleic Acids Res. 16: 7633-7645 (1988) discloses a solution phase approach, using a reagent obtained in situ by treating methylphosphonothioic dichloride with 1-hydroxy-6-trifluoromethyl benzotriazole to introduce a methylphosphonothioate internucleotide linkage into a dinucleotide in 60-70% yield, and produces a hexamer containing the linkage by two consecutive condensations of dimers.
Lebadev et al., Tet. Lett. 31: 855-858 (1990) discloses a solution phase approach to produce dinucleotides containing a stereospecific methylphosphonothioate internucleotide linkage in 50-60% yield.
Stawinski et al., Nucleic Acids Res. Symposium Series No. 21: 47-48 (1989), discloses synthesis of nucleoside H-phosphonothioates and nucleoside methylphosphonothioates.
To use alkylphosphonothioate artificial internucleotide linkages in an antisense approach, however, it is necessary to incorporate such internucleotide linkages into oligonucleotides, rather than dinucleotides. Unfortunately, the related art is devoid of any feasible method for doing this.
Synthesis of oligonucleotides having other non-ionic artificial internucleotide linkages is known in the art. For example, Agrawal and Goodchild, Tet. Lett. 28: 3539-3592 (1987) discloses a nucleoside methylphosphonamidite approach in a standard amidite coupling cycle to produce oligonucleotides having methylphosphonate internucleotide linkages. However, this reference contains no suggestion concerning the synthesis of oligonucleotide methylphosphonothioates or alkylphosphonothioates.
Several references report methods for oxidative sulfurization of oligonucleotides. For example, Stac et al., J. Am. Chem. Soc. 106: 6077-6079 (1984) discloses sulfurization of oligonucleotide phosphite triesters using elemental sulfur in a carbon disulfide:pyridine:triethylamine solution. Beaucage et al., U.S. Pat. No. 5,003,097 (1991) discloses a method for sulfurization of oligonucleotides using 3H-1,2-Benzodithiol-3-one 1,1-dioxide. However, these references demonstrate oxidative sulfurization of natural phosphodiester internucleotide linkages in oligonucleotides and do not demonstrate oxidative sulfurization of an intermediate methylphosphite linkage to generate methylphosphonothiate.
There is, therefore, a need for methods to produce additional modified oligonucleotides having non-ionic artificial internucleotide linkages, such as alkylphosphonothioate or arylphosphonothioate linkages. Ideally, such methods will be adaptable to standard methods for synthesizing oligonucleotides, thereby allowing convenient assembly of the modified oligonucleotides and of chimeric oligonucleotides having varied internucleotide linkages.